Characterization of Ca2+-dependent phospholipase A2 activity during zebrafish embryogenesis

We have developed a simple fluorescent assay for detection of phospholipase A(2) (PLA(2)) activity in zebrafish embryos that utilizes a fluorescent phosphatidylcholine substrate. By using this assay in conjunction with selective PLA(2) inhibitors and Western blot analysis, we identified the principal activity in zebrafish embryogenesis as characteristic of the Ca(2+)-dependent cytosolic PLA(2) (cPLA(2)) subtype. Embryonic cPLA(2) activity remained constant from the 1-cell stage until the onset of somitogenesis, at which time it increased sharply. This increase was preceded by the expression of a previously identified zebrafish cPLA(2) homologue (Nalefski, E., Sultzman, L., Martin, D., Kriz, R., Towler, P., Knopf, J., and Clark, J. (1994) J. Biol. Chem. 269, 18239-18249). By using a quenched BODIPY-labeled phosphatidylcholine that fluoresces only upon cleavage by PLA(2), lipase activity was visualized in the cells of living embryos where it localized to perinuclear membranes.